Difference between revisions of "Part:BBa K3784001"

Revision as of 10:15, 17 October 2021

fldB (Encoding 2-hydroxyacyl-CoA dehydratase protein family)

The fldB is from the FLD gene cluster of Clostridium sporogenes, involved in the conversion of tryptophan to 3-Indolepropionic acid. In the five-step metabolic pathway we reconstructed, fldB encodes subunit of 2-hydroxyacyl-CoA dehydratase protein, combined with another subunit encoded by fldC to become 2-hydroxyacyl-CoA dehydratase protein, which catalyze indole lactic acid to produce indole acrylic acid.

Characterization

We successfully inserted the fldH gene into the His-6p-MBP-RSFD plasmid and transformed it into E. coli BL21(DE3) and induced it to express. After extracting total RNA, we obtained its cDNA by reverse transcription. We used cDNA as a template to amplify the fragment, then checked it by agarose gel electrophoresis. We chose rsmA (16S rRNA m(6)2A1518, m(6)2A1519 dimethyltransferase) as the internal reference gene and set a negative control. The fldB is well expressed and the expression levels of internal reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1169
Illegal EcoRI site found at 1241
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1169
Illegal EcoRI site found at 1241
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1169
Illegal EcoRI site found at 1241
Illegal BglII site found at 48
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1169
Illegal EcoRI site found at 1241
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1169
Illegal EcoRI site found at 1241
1000
COMPATIBLE WITH RFC[1000]

@@ Line 7: / Line 7: @@
 ===Characterization===
-We successfully inserted the fldB gene into the plasmid, which is transcribed by a self-contained promoter cloned from Clostridium sporogenes.
+We successfully inserted the fldH gene into the His-6p-MBP-RSFD plasmid and transformed it into E. coli BL21(DE3) and induced it to express. After extracting total RNA, we obtained its cDNA by reverse transcription. We used cDNA as a template to amplify the fragment, then checked it by agarose gel electrophoresis. We chose rsmA (16S rRNA m(6)2A1518, m(6)2A1519 dimethyltransferase) as the internal reference gene and set a negative control. The fldB is well expressed and the expression levels of internal reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.
-We transformed the plasmid into E. coli BL21(DE3) and induced it to express. After extracting total RNA, we obtained its cDNA by reverse transcription. We used cDNA as a template to amplify the fragment, then checked it by agarose gel electrophoresis. We chose rsmA (16S rRNA m(6)2A1518, m(6)2A1519 dimethyltransferase) as the internal reference gene and set a negative control. The fldB is well expressed and the expression levels of internal reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.
 [[File:BNUZ-fldB.png]]